EP1012186A2 - Keratinocyte-wachstumsfaktor und deren verwendung in zusammensetzung mit derivaten vom gluconähnlichen peptid - Google Patents

Keratinocyte-wachstumsfaktor und deren verwendung in zusammensetzung mit derivaten vom gluconähnlichen peptid

Info

Publication number
EP1012186A2
EP1012186A2 EP97953157A EP97953157A EP1012186A2 EP 1012186 A2 EP1012186 A2 EP 1012186A2 EP 97953157 A EP97953157 A EP 97953157A EP 97953157 A EP97953157 A EP 97953157A EP 1012186 A2 EP1012186 A2 EP 1012186A2
Authority
EP
European Patent Office
Prior art keywords
lys
glu
asn
gly
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP97953157A
Other languages
English (en)
French (fr)
Other versions
EP1012186B1 (de
Inventor
Catherine L. Farrell
Yue-Sheng Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Amgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amgen Inc filed Critical Amgen Inc
Priority to SI9730362T priority Critical patent/SI1012186T1/xx
Publication of EP1012186A2 publication Critical patent/EP1012186A2/de
Application granted granted Critical
Publication of EP1012186B1 publication Critical patent/EP1012186B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]

Definitions

  • the present invention relates to keratinocyte growth factors and to uses thereof .
  • FGFs fibroblast growth factors
  • the fibroblast growth factor (FGF) family is now known to consist of at least fourteen members, namely FGF-1 to FGF-10 and homologous factors FHF-1 to FHF-4, which share a relatedness among primary structures: basic fibroblast growth factor, bFGF
  • FGF-6 Maries et al . (1989), Oncogene, 4:335-340
  • keratinocyte growth factor KGF
  • hisactophilin Habazzettl et al . (1992), Nature, 359:855-858
  • FGF-9 Meyamoto et al . (1993), Mol . Cell Biol . , 13(7) :4251- 4259
  • fibroblast growth factor-10 also known as keratinocyte growth factor-2 , KGF-2 (WO 96/25422).
  • FHFs homologous factors
  • KGF protein product s
  • KGF protein product s
  • These KGF protein product (s) have general applicability and may retain some or all of the biological activity of KGF.
  • Yet another aspect relates to methods of modulating the growth and differentiation of epithelial cells.
  • a patient in need of stimulation (including cytoprotection, proliferation and/or differentiation) of epithelial cells will be administered a therapeutically-effective or prophylactically-effective amounts of a KGF protein product (s) .
  • Figure 1 shows the nucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO: 2) sequences of KGF (the nucleotides encoding the mature form of mature KGF is depicted by bases 96 to 582 of SEQ ID NO:l and the mature form of KGF is depicted by amino acid residues 32 to 194 of SEQ ID NO:2), with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 2 shows the nucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO: 2) sequences of KGF (the nucleotides encoding the mature form of mature KGF is depicted by bases 96 to 582 of SEQ ID NO:l and the mature form of KGF is depicted by amino acid residues 32 to 194 of SEQ ID NO:2), with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 2 shows the nucleotide (SEQ ID NO:
  • amino acid sequences of C(1,15)S amino acid sequences of C(1,15)S, a KGF analog having substitutions of serine for cysteine at amino acid positions 1 and 15 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 3 shows the nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences of ⁇ N3/C(15)S, a KGF analog having a deletion of the first 3 amino acids of the amino-terminus and a substitution of serine for cysteine at amino acid position 15 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 4 shows the nucleotide (SEQ ID NO: 7) and amino acid (SEQ ID NO: 8) sequences of ⁇ N3/C(15)-, a KGF analog having a deletion of the first 3 amino acids of the amino-terminus and a deletion of cysteine at amino acid position 15 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 5 shows the nucleotide (SEQ ID NO: 9) and amino acid (SEQ ID NO: 10) sequences of ⁇ N8/C(15)S, a KGF analog having a deletion of the first 8 amino acids of the amino-terminus and a substitution of serine for cysteine at amino acid position 15 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 6 shows the nucleotide (SEQ ID NO: 9) and amino acid (SEQ ID NO: 10) sequences of ⁇ N8/C(15)S, a KGF analog having a deletion of the first 8 amino acids of the amino-terminus and a substitution of serine for cysteine at amino acid position 15 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 6 shows the nucleotide (SEQ ID NO: 9)
  • Figure 7 shows the nucleotide (SEQ ID NO: 13) and amino acid (SEQ ID NO: 14) sequences of ⁇ N15, a KGF analog having a deletion of the first 15 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 8 shows the nucleotide (SEQ ID NO: 15) and amino acid (SEQ ID NO: 16) sequences of ⁇ N16, a KGF analog having a deletion of the first 16 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 9 shows the nucleotide (SEQ ID NO: 17) and amino acid (SEQ ID NO: 18) sequences of ⁇ N17, a KGF analog having a deletion of the first 17 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 10 shows the nucleotide (SEQ ID NO: 19) and amino acid (SEQ ID NO: 20) sequences of ⁇ N18, a KGF analog having a deletion of the first 18 amino acids of the amino-terminus of KGF, with the initial amino acid being M wherein n equals 0 or 1.
  • Figure 11 shows the nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences of ⁇ N19, a KGF analog having a deletion of the first 19 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 12 shows the nucleotide (SEQ ID NO: 23) and amino acid (SEQ ID NO: 24) sequences of ⁇ N20, a KGF analog having a deletion of the first 20 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 13 shows the nucleotide (SEQ ID NO:25) and amino acid (SEQ ID NO:2 ⁇ ) sequences of ⁇ N21, a KGF analog having a deletion of the first 21 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 14 shows the nucleotide (SEQ ID NO:27) and amino acid (SEQ ID NO:28) sequences of ⁇ N22, a KGF analog having a deletion of the first 22 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 15 shows the nucleotide (SEQ ID NO: 29) and amino acid (SEQ ID NO: 30) sequences of ⁇ N23, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 16 shows the nucleotide (SEQ ID NO:31) and amino acid (SEQ ID NO:32) sequences of ⁇ N24, a KGF analog having a deletion of the first 24 amino acids of the amino-terminus of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 17 shows the nucleotide (SEQ ID NO: 33) and amino acid (SEQ ID NO: 34) sequences of C(l, 15)S/R(144)E, a KGF analog having substitutions of serine for cysteine at amino acid positions 1 and 15 and a substitution of glutamic acid for arginine at amino acid position 144 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 18 shows the nucleotide (SEQ ID NO: 35) and amino acid (SEQ ID NO: 36) sequences of C(l, 15) S/R(144)Q, a KGF analog having substitutions of serine for cysteine at amino acid positions 1 and 15 and a substitution of glutamine for arginine at amino acid position 144 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 19 shows the nucleotide (SEQ ID NO: 35) and amino acid (SEQ ID NO: 36) sequences of C(l, 15) S/R(144)Q, a KGF analog having substitutions of serine for cysteine at amino acid positions 1 and 15 and a substitution of glutamine for arginine at amino acid position 144 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 19 shows the nucleotide (SEQ ID NO: 35) and amino acid (SEQ ID NO: 36) sequences of C(l
  • Figure 20 shows the nucleotide (SEQ ID NO: 39) and amino acid (SEQ ID NO: 40) sequences of C (1,15, 40) S, a KGF analog having substitutions of serine for cysteine at amino acid positions 1, 15 and 40 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 21 shows the nucleotide (SEQ ID NO: 41) and amino acid (SEQ ID NO: 42) sequences of C(l, 15, 102) S, a KGF analog having substitutions of serine for cysteine at amino acid positions 1, 15 and 102 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 22 shows the nucleotide (SEQ ID NO: 43) and amino acid (SEQ ID NO: 44) sequences of
  • Figure 24 shows the nucleotide (SEQ ID NO: 47) and amino acid (SEQ ID NO: 48) sequences of ⁇ N23/K(139)E, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamic acid for lysine at amino acid position 139 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 25 shows the nucleotide (SEQ ID NO: 49) and amino acid (SEQ ID NO: 50) sequences of ⁇ N23/K(139)Q, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamine for lysine at amino acid position 139 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 26 shows the nucleotide (SEQ ID NO: 49) and amino acid (SEQ ID NO: 50) sequences of ⁇ N23/K(139)Q, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamine for lysine at amino acid position 139 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 26 shows the nucleotide (SEQ ID NO: 49) and amino acid (SEQ ID NO: 50) sequences of ⁇ N23/K(139)Q,
  • Figure 27 shows the nucleotide (SEQ ID NO: 53) and amino acid (SEQ ID NO: 54) sequences of ⁇ N23/R(144)E, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamic acid for arginine at amino acid position 144 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 28 shows the nucleotide (SEQ ID NO: 55) and amino acid (SEQ ID NO: 56) sequences of ⁇ N23/R(144)L, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of leucine for arginine at amino acid position 144 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 29 shows the nucleotide (SEQ ID NO: 57) and amino acid (SEQ ID NO: 58) sequences of ⁇ N23/K(147)E, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamic acid for lysine at amino acid position 147 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 30 shows the nucleotide (SEQ ID NO: 59) and amino acid (SEQ ID NO: 60) sequences of ⁇ N23/K(147)Q, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamine for lysine at amino acid position 147 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 31 shows the nucleotide (SEQ ID NO: 59) and amino acid (SEQ ID NO: 60) sequences of ⁇ N23/K(147)Q, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamine for lysine at amino acid position 147 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 31 shows the nucleotide (SEQ ID NO: 59) and amino acid (SEQ ID NO: 60) sequences of ⁇ N23/K(147
  • Figure 32 shows the nucleotide (SEQ ID NO: 63) and amino acid (SEQ ID NO: 64) sequences of ⁇ N23/K(153)Q, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamine for lysine at amino acid position 153 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • Figure 33 shows the nucleotide (SEQ ID NO: 65) and amino acid (SEQ ID NO: 66) sequences of ⁇ N23/Q(152)E/K(153)E, a KGF analog having a deletion of the first 23 amino acids of the amino-terminus and a substitution of glutamic acid for glutamine at amino acid position 152 of KGF and glutamic acid for lysine at amino acid position 153 of KGF, with the initial amino acid being M n wherein n equals 0 or 1.
  • KGF protein is meant the protein defined by amino acids Cys 32 to Thr 194 of SEQ ID NO: 2 (mature KGF) and variant proteins thereof.
  • amino acid numbering for molecules described herein shall correspond to that presented for the mature form of molecule (i.e., minus the signal sequence) , as depicted by amino acids 32 to 194 of SEQ ID NO: 2, with the initial MET in each such sequence being residue number "0”.
  • KGF protein (s) thus includes a protein in which one or more amino acid residues have been deleted from (“deletion varian (s) ”) , inserted into (“addition variant (s) ”) , and/or substituted for (“substitution variant (s)”) residues within the amino acid sequence of SEQ ID NO: 2 and which retains biological activity.
  • biological activity means that a KGF protein (s) possesses some but not necessarily all the same properties of (and not necessarily to the same degree as) KGF. The selection of the particular properties of interest depends upon the desired use of the desired KGF protein(s).
  • amino acid sequence variant There are two principal variables in the construction of amino acid sequence variant (s): the location of the mutation site and the nature of the mutation. In designing each variant (s), the location of each mutation site and the nature of each mutation will depend on the biochemical characteristic (s) to be modified. Each mutation site can be modified individually or in series, e.g., by (1) deleting the target amino acid residue, (2) inserting one or more amino acid residues adjacent to the located site or (3) substituting first with conservative amino acid choices and, depending upon the results achieved, then with more radical selections.
  • Amino acid sequence deletions generally range from about 1 to 30 amino acid residues, preferably from about 1 to 20 amino acids, more preferably from about 1 to 10 amino acid residues and most preferably from about 1 to 5 residues. Amino-terminal, carboxy- terminal and internal intrasequence deletions are contemplated. Deletions within the amino acid sequence of KGF may be made, for example, in regions of low homology with the sequences of other members of the FGF family. Deletions within the amino acid sequence of
  • KGF in areas of substantial homology with the sequences of other members of the FGF family will be more likely to significantly modify the biological activity.
  • the number of total deletions and/or consecutive deletions preferably will be selected so as to preserve the tertiary structure of KGF in the affected domain, e.g., cysteine crosslinking.
  • An amino acid sequence addition may include insertions of an amino- and/or carboxyl-terminal fusion ranging in length from one residue to one hundred or more residues, as well as internal intrasequence insertions of single or multiple amino acid residues. Internal additions may range generally from about 1 to 20 amino acid residues, preferably from about 1 to 10 amino acid residues, more preferably from about 1 to 5 amino acid residues, and most preferably from about 1 to 3 amino acid residues . Additions within the amino acid sequence of KGF may be made in regions of low homology with the sequences of other members of the FGF family. Additions within the amino acid sequence of KGF in areas of substantial homology with the sequences of other members of the FGF family will be more likely to significantly modify the biological activity. Insertions or additions preferably include amino acid sequences derived from the sequences of other FGF family members.
  • amino-terminus addition is contemplated to include the addition of a methionine (for example, as an artifact of the direct expression in bacterial recombinant cell culture) or an amino acid residue or sequence of KGF.
  • a further example of an amino-terminal addition includes the fusion of a signal sequence to the amino-terminus of KGF in order to facilitate the secretion of protein from recombinant host cells. Such signal sequences generally will be obtained from and thus be homologous to the intended host cell species.
  • the signal sequence may be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase or heat- stable enterotoxin II leader sequences.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase or heat- stable enterotoxin II leader sequences.
  • each polypeptide may have a signal sequence selected, for example, from the group of the yeast invertase, alpha factor or acid phosphatase leader sequences.
  • signal sequences of KGF are satisfactory, although other mammalian signal sequences may be suitable, for example sequences derived from other FGF family members may be suitable.
  • an amino- or a carboxy-terminus addition includes chimeric proteins comprising the amino-terminal or carboxy-terminal fusion of a KGF protein with all or part of the constant domain of the heavy or light chain of human immunoglobulin (individually or collectively, ("KGF Fc(s)").
  • KGF Fc(s) chimeric polypeptides are preferred wherein the immunoglobulin portion of each comprises all of the domains except the first domain of the constant region of the heavy chain of human immunoglobulin such as IgG (e.g., IgGl or IgG3 ) , IgA, IgM or IgE.
  • any amino acid of the immunoglobulin portion can be deleted or substituted with one or more amino acids, or one or more amino acids can be added as long as the KGF protein portion is capable of stimulating epithelial cells and the immunoglobulin portion shows one or more of its characteristic properties.
  • variants are amino acid substitution variant (s) of the amino acid sequence of KGF. These are variant (s) wherein at least one amino acid residue in KGF is removed and a different residue inserted in its place.
  • substitution variant (s) include allelic variant (s) , which are characterized by naturally-occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change.
  • allelic variant (s) are characterized by naturally-occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change.
  • One skilled in the art can use any information known about the binding or active site of the polypeptide in the selection of possible mutation sites .
  • alanine scanning mutagenesis One method for identifying amino acid residues or regions for mutagenesis of a protein is called “alanine scanning mutagenesis", as described by Cunningham and Wells (1989), Science, 244:1081-1085, the disclosure of which is hereby incorporated by reference.
  • an amino acid residue or group of target residues of a protein is identified (e.g., charged residues such as Arg, Asp, His, Lys and Glu) and replaced by a neutral or negatively-charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains/residues demonstrating functional sensitivity to the substitutions are then refined by introducing additional or alternate residues at the sites of substitution.
  • the site for introducing an amino acid sequence modification is predetermined.
  • alanine scanning or random mutagenesis may be conducted and the variant (s) may be screened for the optimal combination of desired activity and degree of activity.
  • the sites of greatest interest for substitutional mutagenesis include sites in which particular residues within KGF are substantially different from various species or other FGF family members in terms of side-chain bulk, charge, and/or hydrophobicity.
  • sites of interest include those in which particular residues within KGF are identical among various species or other FGF family members, as such positions are generally important for the biological activity of a protein. A skilled artisan will appreciate that initially these sites should be modified by substitution in a relatively conservative manner .
  • substitutions may be introduced and/or other additions/deletions may be made and the resulting polypeptides screened.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle (1982), J “ . Mol . Biol . , 157:105-131, the disclosure of which is incorporated herein by reference) . It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the functionally equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. U.S.
  • Patent 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
  • U.S. Patent 4,554,101 also teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophilicity. Through the methods disclosed in U.S. Patent 4,554,101 a skilled artisan would be able to identify epitopes, for example, within the amino acid sequence of KGF. These regions are also referred to as "epitopic core regions". Numerous scientific publications have been devoted to the prediction of secondary structure, and to the identification of epitopes, from analyses of amino acid sequences (Chou and Fasman (1974) , Biochemistry, 13 (2) :222-245; Chou and Fasman (1974), Biochemistry, 13 (2) : 211-222;, Chou and Fasman (1978), Adv. Enzymol .
  • KGF KGF-like growth factor
  • substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the relative charge or hydrophobicity of the protein at the target site or (c) the bulk of the side chain.
  • Naturally-occurring residues are divided into groups based on common side chain properties :
  • Non-conservative substitutions may involve the exchange of a member of one of these groups for another.
  • Such substituted residues may be introduced into regions of KGF that, for example, are homologous with other FGF family members or into non-homologous regions of the protein.
  • a variety of amino acid substitutions or deletions may be made to modify or add N-linked or O-linked glycosylation sites, resulting in a protein with altered glycosylation.
  • the sequence may be modified to add glycosylation sites to or to delete
  • N-linked or O-linked glycosylation sites from the KGF proteins N-linked or O-linked glycosylation sites from the KGF proteins.
  • An asparagine-linked glycosylation recognition site comprises a tripeptide sequence which is specifically recognized by appropriate cellular glycosylation enzymes . These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro.
  • Specific mutations of the sequences of KGF may involve substitution of a non-native Asn at position 50 and Thr at position 52 to insert an N-linked glcosylation site. Such modifications may be of particular utility in the addition of a carbohydrate, which may reduce aggregation (to increase thermal and storage stability) , increase half-life in circulation, decrease isoelectric point, increase solubility and decrease antigenecity of the protein.
  • a variant polypeptide will preferably be substantially homologous to the amino acid sequence of KGF (SEQ ID NO: 2) .
  • the term "substantially homologous” as used herein means a degree of homology that is in excess of 80%, preferably in excess of 90%, more preferably in excess of 95% or most preferably even 99%.
  • the percentage of homology as described herein is calculated as the percentage of amino acid residues found in the smaller of the two sequences which align with identical amino acid residues in the sequence being compared when four gaps in a length of 100 amino acids may be introduced to assist in that alignment, as set forth by Dayhoff (1972), Atlas of Protein Sequence and Structure, 5_:124, National Biochemical Research Foundation, Washington, D.C., the disclosure of which is hereby incorporated by reference. Also included within the term “substantially homologous" are variant (s) of KGF which may be isolated by virtue of cross-reactivity with antibodies to amino acids sequence of SEQ ID NO: 2, or whose genes may be isolated through hybridization with the DNA of SEQ ID N0:1 or with segments thereof.
  • a KGF protein may be rapidly screened to assess its physical properties.
  • the level of biological activity e.g., receptor binding and/or affinity, mitogenic, cell proliferative and/or in vivo activity
  • the level of biological activity may be tested using a variety of assays.
  • One such assay includes a mitogenic assay to test the ability of a protein to stimulate DNA synthesis (Rubin et al . (1989), supra, the disclosure of which is hereby incorporated by reference) .
  • Another such assay includes a cell proliferative assay to test the ability of a protein to stimulate cell proliferation (Falco et al . (1988), Oncogene, 2.:573- 578, the disclosure of which is hereby incorporated by reference) .
  • KGF KGF-like growth factor
  • WO 9008771, WO 9622369, WO 9611949 and WO 9611951 the disclosures of which are hereby incorporated by reference.
  • KGF includes proteins having residues corresponding to Cys 1 and Cys 15 of SEQ ID NO: 2 replaced or deleted, with the resultant molecule having improved stability as compared with the parent molecule (WO 96/11949) .
  • KGF includes charge- change polypeptides wherein one or more of amino acid residues 41-154 of native KGF (preferably residues Arg 41 , Gin 43 , Lys 55 , Lys 95 , Lys 128 , Asn 137 , Gin 138 , Lys 139 , Arg 144 , Lys 147 , Gin 152 , Lys 153 or Thr 154 ) are deleted or substituted with a neutral residue or 8/24813
  • amino acid residues 41-154 of native KGF preferably residues Arg 41 , Gin 43 , Lys 55 , Lys 95 , Lys 128 , Asn 137 , Gin 138 , Lys 139 , Arg 144 , Lys 147 , Gin 152 , Lys 153 or Thr 154
  • KGF includes proteins generated by substituting at least one amino acid having a higher loop-forming potential for at least one amino acid within a loop-forming region of Asn 115 -His 11 ⁇ -Tyr 117 - Asn 118_ ⁇ hr 119 of native KGF (WO 96/11950) .
  • a still yet further example includes proteins having one or more amino acid substitutions, deletions or additions within a region of 123-133 (amino acids 154-164 of SEQ ID NO: 2) of native KGF; these proteins may have agonistic or antagonistic activity.
  • proteins include the following: C(1,15)S ( Figure 2); ⁇ N3/C(15)S ( Figure 3 ) ; ⁇ N3/C(15)- ( Figure 4); ⁇ N8/C(15)S (Figure 5) ; ⁇ N8/C(15)- ( Figure 6); ⁇ N15 ( Figure 7); ⁇ N16 ( Figure 8); ⁇ N17 ( Figure 9); ⁇ N18 ( Figure 10); ⁇ N19 ( Figure 11); ⁇ N20 ( Figure 12); ⁇ N21 ( Figure 13); ⁇ N22 ( Figure 14); ⁇ N23 ( Figure 15); ⁇ N24 ( Figure 16); C(1,15)S/R(144)E ( Figure 17); C (1, 15) S/R (144) Q
  • derivatives Chemically-modified derivatives of KGF protein (s) in which the protein is linked to a polymer in order to modify properties of the protein (referred herein as "derivatives") are included within the scope of the present invention. Such derivatives may be prepared by one skilled in the art given the disclosures herein. Conjugates may be prepared using glycosylated, non-glycosylated or de-glycosylated KGF proteins and suitable chemical moieties. Typically non-glycosylated proteins and water soluble polymers will be used.
  • Water soluble polymers are desirable because the protein to which each is attached will not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition.
  • One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically and, if so, the therapeutic profile of the protein (e.g., duration of sustained release; resistance to proteolysis; effects, if any, on dosage; biological activity; ease of handling; degree or lack of antigenicity and other known effects of a water soluble polymer on a therapeutic protein) .
  • Suitable, clinically acceptable, water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , polyethylene glycol propionaldehyde, copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol (PVA) , polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3 , 6-trioxane, ethylene/maleic anhydride copolymer, poly ( ⁇ -amino acids) (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers (PPG) and other polyalkylene oxides, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other polyoxyethylated polyols
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the water soluble polymers each may be of any molecular weight and may be branched or unbranched. Generally, the higher the molecular weight or the more branches, the higher the polymer : protein ratio.
  • the water soluble polymers each typically have an average molecular weight of between about 2kDa to about lOOkDa (the term "about” indicating that in preparations of a water soluble polymer, some molecules will weigh more, some less, than the stated molecular weight) .
  • the average molecular weight of each water soluble polymer preferably is between about 5kDa and about 40kDa, more preferably between about lOkDa and about 35kDa and most preferably between about 15kDa and about 30kDa.
  • a specific embodiment of the present invention is an unbranched monomethoxy-polyethylene glycol aldehyde molecule having an average molecular weight of either about 20kDa or aboput 33kDa (e.g., between 30kDa and 35kDa) , or a tertiary butyl polyethylene glycol additive having an average molecular weight of about 33kDa (e.g., between 30kDa and 35kDa) conjugated via reductive alkylation to the KGF protei (s) .
  • Polyvalent forms i.e., molecules comprising more than one active moiety, may be constructed.
  • the molecule may possess multiple KGF protein(s). Additionally, the molecule may possess at least one KGF protein (s) and, depending upon the desired characteristic of polyvalent form, at least one other molecule.
  • the polyvalent form may be constructed, for example, by chemically coupling at least one KGF protein (s) and another moeity with any clincally accepted linker (e.g., a water soluble polymer) .
  • linker e.g., a water soluble polymer
  • the linker must not impart new immunogenecity nor, by virtue of the new amino acid residues, alter the hydrophobicity and charge balance of the structure which affects its biodistribution and clearance.
  • the water soluble polymers can be, based on the monomers listed above, homopolymers, random or block copolymers, terpolymers straight chain or branched, substituted or unsubstituted.
  • the polymer can be of any length or molecular weight, but these characteristics can affect the biological properties. Polymer average molecular weights particularly useful for decreasing clearance rates in pharmaceutical applications are in the range of 2,000 to 35,000 daltons.
  • the length of the polymer can be varied to optimize or confer the desired biological activity.
  • the active moieties may be linked using conventional coupling techniques (see WO 2011/001100600A1100A1100A1100A1100A1100A1100A1100
  • a bivalent molecule may consist of two tandem repeats of KGF protein (s) separated by a polypeptide linker region.
  • the design of the polypeptide linkers is similar in design to the insertion of short loop sequences between domains in the de novo design of proteins (Mutter (1988) , TIBS, 11:260-265 and Regan and DeGrado (1988), Science, 241:976-978, the disclosures of which are hereby incorporated by reference) .
  • linker constructs have been assembled and shown to be useful for forming single chain antibodies; the most functional linkers vary in size from 12 to 25 amino acids (amino acids having unreactive side groups, e.g., alanine, serine and glycine) which together constitute a hydrophilic sequence, have a few oppositely charged residues to enhance solubility and are flexible (Whitlow and Filpula (1991) , Methods : A Companion to Methods in Enzymology, 2:97-105; and Brigido et al . (1993), J " . Immunol . , 150:469-479, the disclosures of which are hereby incorporated by reference) .
  • amino acids having unreactive side groups e.g., alanine, serine and glycine
  • KGF protein (s) may be chemically coupled to a biotin, and resulting conjugate is then allowed to bind to avidin, resulting in tetravalent avidin/biotin/KGF protein (s).
  • KGF protein (s) may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates.
  • DNP dinitrophenol
  • TNP trinitrophenol
  • recombinant fusion proteins may also be produced wherein each recombinant chimeric molecule has a KGF protein sequence amino- terminally or carboxy- erminally fused to all or part of the constant domains, but at least one constant domain, of the heavy or light chain of human immunoglobulin.
  • a chimeric KGF protein/IgGl (or IgGl/KGF protein) fusion protein may be produced from a light chain-containing chimeric gene: a KGF protein/human kappa light chain chimera (KGF protein/Ck) or a human kappa light chain/KGF protein chimera (Ck/KGF protein) ; or a heavy chain- containing chimeric gene: a KGF protein/human gamma-1 heavy chain chimera (KGF protein/Cg-1) or a human gamma-1 heavy chain/KGF protein chimera (Cg-l/KGF protein) .
  • the gene products may be assembled into a single chimeric molecule having a KGF protein (s) displayed bivalently. Additional details relating to the construction of such chimeric molecules are disclosed in United States Patent 5,116,964, WO 89/09622, WO 91/16437 and EP 315062, the disclosures of which are hereby incorporated by reference.
  • recombinant fusion proteins may also be produced wherein each recombinant chimeric molecule has at least one KGF protein (s) , as described above, and at least a portion of the region 186-401 of osteoprotegerin (OPG) , as described in European Patent Application No. 96309363.8, the disclosure of which is hereby incorporated by reference.
  • KGF protein (s) or the portion of osteoprotogerin may be at the amino- terminus or the carboxy-terminus of the chimeric molecule .
  • KGF protein (s) is described in further detail below.
  • Such proteins may be prepared, for example, by recombinant techniques or by in vi tro chemical synthesis of the desired KGF protein (s) .
  • polynucleotides Based upon the present description and using the universal codon table, one of ordinary skill in the art can readily determine all of the nucleic acid sequences which encode the amino acid sequence of KGF protein (s) . Recombinant expression techniques conducted in accordance with the descriptions set forth below may be followed to produce these polynucleotides and to express the encoded proteins. For example, by inserting a nucleic acid sequence which encodes a KGF protein (s) into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding a KGF protein (s) can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the desired protein (s) may be produced in large amounts .
  • host/vector systems available for the propagation of desired nucleic acid sequences and/or the production of the desired protein. These include, but are not limited to, plasmid, viral and insertional vectors, and prokaryotic and eukaryotic hosts.
  • plasmid plasmid
  • viral and insertional vectors plasmid
  • prokaryotic and eukaryotic hosts prokaryotic and eukaryotic hosts.
  • One skilled in the art can adapt a host/vector system which is capable of propagating or expressing heterologous DNA to produce or express the sequences of the present invention.
  • nucleic acid sequences within the scope of the present invention include the nucleic acid sequence of Figure 1, as well as degenerate nucleic acid sequences thereof, nucleic acid sequences which encode variant (s) of KGF and those nucleic acid sequences which hybridize (under hybridization conditions disclosed in the cDNA library screening section below, or equivalent conditions or more stringent conditions) to complements of the nucleic acid sequence of Figure 1.
  • nucleic acid sequences encoding a desired protein is in operative association with a suitable expression control or regulatory sequence capable of directing the replication and/or expression of the desired protein in a selected host.
  • a nucleic acid sequence encoding a KGF protein can readily be obtained in a variety of ways including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening and/or PCR amplification of cDNA. These methods and others which are useful for isolating such nucleic acid sequences are set forth in Sambrook et al . (1989) , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; by Ausubel et al . (1994), Current Protocols in
  • nucleic acid sequence synthesis methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid sequence synthesis.
  • Large nucleic acid sequences for example those larger than about 100 nucleotides in length, can be synthesized as several fragments . The fragments can then be ligated together to form a suitable nucleic acid sequence.
  • a preferred method is polymer-supported synthesis using standard phosphoramidite chemistry.
  • a suitable nucleic acid sequence may be obtained by screening an appropriate cDNA library (i.e., a library prepared from one or more tissue or cell sources believed to express the protein) or a genomic library (a library prepared from total genomic DNA) .
  • the source of the cDNA library is typically a tissue from any species that is believed to express a desired protein in reasonable quantities.
  • the source of the genomic library may be any tissue or tissues from any mammalian or other species believed to harbor a gene encoding a desired protein.
  • Hybridization media can be screened for the presence of a DNA encoding a desired protein using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the cDNA or gene to be cloned) that will hybridize selectively with cDNA(s) or gene(s) present in the library.
  • the probes typically used for such screening encode a small region of DNA sequence from the same or a similar species as the species from which the library is prepared. Alternatively, the probes may be degenerate, as discussed herein.
  • Hybridization is typically accomplished by annealing an oligonucleotide probe or cDNA to the clones under conditions of stringency that prevent non-specific binding but permit binding of those clones that have a significant level of homology with the probe or primer.
  • Typical hybridization and washing stringency conditions depend in part on the size (i.e., number of nucleotides in length) of the cDNA or oligonucleotide probe and whether the probe is degenerate. The probability of identifying a clone is also considered in designing the hybridization medium (e.g., whether a cDNA or genomic library is being screened) .
  • hybridization conditions include those as set forth in Ausubel et al . (1994), supra .
  • the hybridization medium is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, the hybridization medium being screened, the number of clones being screened and the like.
  • Exemplary stringent hybridization conditions are hybridization in 6 x SSC at 62-67°C, followed by washing in 0.1 x SSC at 62-67°C for approximately one hour.
  • exemplary stringent hybridization conditions are hybridization at 45-55% formamide, 6 x SSC at 40-45°C, followed by washing in 0.1 x SSC at 62- 67°C for approximately one hour.
  • Examples of such relaxed stringency hybridization conditions are 6 x SSC at 45-55°C or hybridization with 30-40% formamide at 40-45°C, followed by washing in 1-2 x SSC at 55°C for approximately 30 minutes. See Maniatis et al. (1982), Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages 387 to 389, the disclosure of which is hereby incorporated by reference.
  • oligonucleotide probes are used to screen hybridization media.
  • a first protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate at a temperature of between about 35°C and 63 °C, depending on the length of the probe. For example, 14 base probes are washed at 35-40°C, 17 base probes at 45-50°C, 20 base probes at 52-57°C, and 23 base probes at 57-63°C. The temperature can be increased 2-3°C where the background non-specific binding appears high.
  • a second protocol uses tetramethylammonium chloride (TMAC) for washing.
  • TMAC tetramethylammonium chloride
  • One such stringent washing solution is 3 M TMAC, 50mM Tris-HCl, pH 8.0 and 0.2% SDS.
  • PCR polymerase chain reaction
  • cDNA is prepared from poly(A)+RNA or total RNA using the enzyme reverse transcriptase.
  • Two primers typically complementary to two separate regions of cDNA (oligonucleotides) encoding the desired protein, are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers .
  • the oligonucleotide sequences selected as probes or primers should be of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur during screening or PCR amplification.
  • the actual sequence of the probes or primers is usually based on conserved or highly homologous sequences or regions.
  • the probes or primers can be fully or partially degenerate, i.e., can contain a mixture of probes/primers, all encoding the same amino acid sequence but using different codons to do so.
  • An alternative to preparing degenerate probes is to place an inosine in some or all of those codon positions that vary by species.
  • the oligonucleotide probes or primers may be prepared by chemical synthesis methods for DNA, as described herein.
  • Vectors DNA encoding the desired protein may be inserted into vectors for further cloning (amplification of the DNA) or for expression. Suitable vectors are commercially available or may be specifically constructed. The selection or construction of an appropriate vector will depend on (1) whether it is to be used for DNA amplification or for DNA expression, (2) the size of the DNA to be inserted into the vector and (3) the intended host cell to be transformed with the vector.
  • the vectors each typically involve a nucleic acid sequence which encodes a desired protein operatively linked to one or more of the following expression control or regulatory sequences capable of directing, controlling or otherwise effecting the expression of a desired protein by a selected host cell.
  • Each vector contains various components, depending on its function (amplification of DNA or expression of DNA) and its compatibility with the intended host cell.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selection or marker genes, a promoter, an enhancer element, a transcription termination sequence and the like. These components may be obtained from natural sources or be synthesized by known procedures.
  • suitable prokaryotic cloning vectors include bacteriophages such as lambda derivatives, or plasmids from E. coli (e.g. pBR322, col El, pUC, the F-factor and Bluescript® plasmid derivatives (Stratagene, LaJolla, CA) ) .
  • Other appropriate expression vectors of which numerous types are known in the art for the host cells described herein, can also be used for this purpose.
  • the nucleic acid encoding a signal sequence may be inserted 5' of the sequence encoding a desired protein, e.g, it may be a component of a vector or it may be a part of a nucleic acid encoding a desired protein.
  • the nucleic acid encoding the native signal sequence of KGF is known (WO 90/08771) .
  • Expression and cloning vectors each generally include a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is typically one that enables the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or autonomously replicating sequence.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria, and various origins (e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter) .
  • the expression and cloning vectors each typically contain a selection gene. This gene encodes a "marker" protein necessary for the survival or growth of the transformed host cells when grown in a selective culture media. Host cells that are not transformed with the vector will not contain the selection gene and, therefore, will not survive in the culture media.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline; (b) complement auxotrophic deficiencies or (c) supply critical nutrients not available from the culture media.
  • selection genes may be used to amplify the genes to be expressed. Amplification is the process wherein genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells .
  • suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase.
  • DHFR selection gene cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture media that contains methotrexate, a competitive antagonist of DHFR.
  • An appropriate host cell when wild-type DHFR is used is the Chinese hamster ovary cell line deficient in DHFR activity (Urlaub and Chasin (1980), Proc . Natl . Acad.
  • the transformed cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of other DNA present in the expression vector, such as the DNA encoding a desired protein.
  • Expression and cloning vectors each will typically contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid sequence encoding the desired protein.
  • a promoter is an untranslated sequence located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription and translation of a particular nucleic acid sequence.
  • a promoter may be conventionally grouped into one of two classes, inducible promoters and constitutive promoters.
  • An inducible promoter initiates increased levels of transcription from DNA under its control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
  • a large number of promoters, recognized by a variety of potential host cells, are well known.
  • a promoter may be operably linked to DNA encoding a desired protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence.
  • the native KGF promoter sequence may be used to direct amplification and/or expression of the DNA encoding a desired protein.
  • a heterologous promoter is preferred, however, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter and if it is compatible with the host cell system that has been selected for use.
  • any one of the native promoter sequences of other FGF family members may be used to direct amplification and/or expression of the DNA encoding a desired protein.
  • Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; a bacterial luminescence (luxR) gene system and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their nucleotide sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence (s) using linkers or adaptors as needed to supply any required restriction sites.
  • Suitable promoter sequences for use with yeast hosts are also well known in the art.
  • Suitable promoters for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and, most preferably, Simian Virus 40 (SV40) .
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and, most preferably, Simian Virus 40 (SV40) .
  • Other suitable mammalian promoters include heterologous mammalian promoters, e.g
  • Enhancer Element The expression and cloning vectors each will typically contain an enhancer sequence to increase the transcription by higher eukaryotes of a DNA sequence encoding a desired protein. Enhancers are cis-acting elements of DNA, usually from about 10-300 bp in length, that act on the promoter to increase its transcription.
  • Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Yeast enhancers are advantageously used with yeast promoters. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto- protein and insulin) . Additionally, viral enhancers such as the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into a vector at a position 5 ' or 3 ' to a DNA encoding a desired protein, it is typically located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells each will typically contain a sequence necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5 ' and occasionally 3 ' untranslated regions of eukaryotic DNAs or cDNAs . These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding a desired protein.
  • a suitable vector containing one or more of the above-listed components may be accomplished by standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored and religated in the desired order to generate the required vector. To confirm that the correct sequence has been constructed, the ligation mixture may be used to transform E. coli , and successful transformants may be selected by known techniques as described herein. Quantities of the vector from the transformants are then prepared, analyzed by restriction endonuclease digestion and/or sequenced to confirm the presence of the desired construct.
  • a vector that provides for the transient expression of DNA encoding a desired protein in mammalian cells may also be used.
  • transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of the desired protein encoded by the expression vector.
  • Each transient expression system comprising a suitable expression vector and a host cell, allows for the convenient positive identification of proteins encoded by cloned DNAs as well as for the rapid screening of such proteins for desired biological or physiological properties.
  • prokaryotic and eukaryotic host cells include bacterial, mammalian, fungal, insect, yeast or plant cells.
  • Prokaryotic host cells include, but are not limited to, eubacteria such as Gram-negative or Gram- positive organisms (e.g., E. coli (HB101, DH5a, DH10, and MC1061) ; Bacilli spp. such as B. subtilis; Pseudomonas spp. such as P. aeruginosa; Streptomyces spp.; Salmonella spp. such as S. typhimurium; or Serratia spp. such as S. marcescans .
  • a desired protein may be expressed in E. coli .
  • a KGF protein may be expressed in glycosylated form by any one of a number of suitable host cells derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture might be used, whether such culture involves vertebrate or invertebrate cells, including plant and insect cells.
  • Eukaryotic microbes such as filamentous fungi or yeast may be suitable hosts for the expression of a desired protein.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms, but a number of other genera, species and strains are well known and commonly available.
  • Vertebrate cells may be used, as the propagation of vertebrate cells in culture (tissue culture) is a well-known procedure.
  • useful mammalian host cell lines include, but are not limited to, monkey kidney CVl line transformed by SV40 (COS-7) , human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture) , baby hamster kidney cells and Chinese hamster ovary cells.
  • Other suitable mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, and BHK or HaK hamster cell lines.
  • a desired protein may be expressed in COS cells or in baculovirus cells.
  • a host cell may be transfected and preferably transformed with a desired nucleic acid under appropriate conditions permitting expression of the nucleic acid.
  • suitable host cells and methods for transformation, culture, amplification, screening and product production and purification are well known in the art (Gething and Sambrook (1981) , Nature, 293 : 620-625 or, alternatively, Kaufman et al . (1985), Mol . Cell . Biol . , 5(7) :1750-1759. or U.S. Pat. No. 4,419,446, the disclosures of which are hereby incorporated by reference) .
  • the calcium phosphate precipitation method may be used for mammalian cells without cell walls. Electroporation, microinjection and other known techniques may also be used.
  • a desired protein may be produced by homologous recombination or with recombinant production methods utilizing control elements introduced into cells already containing DNA encoding a KGF protein (s).
  • Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes (Kucherlapati (1989), Prog, in Nucl . Acid Res . and Mol . Biol . , 3_6:301, the disclosure of which is hereby incorporated by reference) .
  • the basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al .
  • each of the one or more recombinant host cells for production of a desired protein will vary depending upon many factors and considerations; the optimum production procedure for a given situation will be apparent to those skilled in the art through minimal experimentation.
  • Such recombinant host cells are cultured in suitable media and the expressed protein is then optionally recovered, isolated and purified from the culture media (or from the cell, if expressed intracellularly) by appropriate means known to those skilled in the art.
  • each of the recombinant cells used to produce a desired protein may be cultured in media suitable for inducing promoters, selecting suitable recombinant host cells or amplifying the gene encoding the desired protein.
  • the media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics (such as gentamicin) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or another energy source.
  • hormones and/or other growth factors such as insulin, transferrin or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium and phosphate
  • buffers such as HEPES
  • nucleosides such as adenosine and thym
  • Suitable culture conditions such as temperature, pH and the like, are also well known to those skilled in the art for use with the selected host cells.
  • the resulting expression product may then be purified to near homogeneity using procedures known in the art. Exemplary purification techniques are taught in WO 90/08771 and WO 96/11952, the disclosures of which are hereby incorporated by reference.
  • KGF protein (s) and chemically-modified derivatives thereof having biological activity may be used as research reagents and as therapeutic and diagnostic agents.
  • a KGF protein product (s) may be used in in vi tro and/or in vivo diagnostic assays to quantify the amount of KGF in a tissue or organ sample.
  • a KGF protein product (s) can be used for identification of the receptor (s) for KGF protein (s) in various body fluids and tissue samples using techniques known in the art (WO 90/08771) .
  • This invention also contemplates the use of a KGF protein product (s) in the generation of antibodies made against the KGF protein product (s), including KGF.
  • KGF protein product s
  • One of ordinary skill in the art can use well-known, published procedures to obtain monoclonal, polyclonal antibodies or recombinant antibodies. Such antibodies may then be used to purify and characterize the KGF protein product (s) .
  • the present invention encompasses pharmaceu- tical preparations each containing therapeutically- or prophylatically-effective amounts of a KGF protein product (s) .
  • compositions each will generally include a therapeutically-effective or prophylatically-effective amount of a KGF protein product (s) in admixture with a vehicle.
  • the vehicle preferably includes one or more pharmaceutically and physiologically acceptable formulation materials in admixture with the KGF protein product (s).
  • the primary solvent in a vehicle may be either aqueous or non-aqueous in nature.
  • the vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH, preferably to maintain the the pH between about 6.5 and 7.0 (e.g., buffers such as citrates, phosphates, and amino acids such as glycine); osmolarity (e.g., mannitol and sodium chloride) ; viscosity; clarity; color; sterility; stability (e.g., sucrose and sorbitol); odor of the formulation; rate of dissolution (e.g., solubilizers or solubilizing agents such as alcohols, polyethylene glycols and sodium chloride) ; rate of release; as well as bulking agents for lyophilized formulation (e.g., mannitol and glycine); surfactants (e.g., polysorbate 20, polysorbate 80, triton, and pluronics) ; antioxidants (e.g
  • the composition may also involve particulate preparations of polymeric compounds such as bulk erosion polymers (e.g., poly (lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic and glycolic acid, poly (cyanoacrylates) ) ; surface erosion polymers (e.g., poly (anhydrides) and poly(ortho esters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl alcohol), poly (vinylpyrrolidone) , maleic anhydride- alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin, and starches and dextrans) and composition systems thereof; or preparations of liposomes or microspheres .
  • bulk erosion polymers e.g., poly (lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives.
  • the optimal pharmaceutical formulation for a desired protein will be determined by one skilled in the art depending upon the route of administration and desired dosage. Exemplary pharmaceutical compositions are disclosed in Remington 's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, PA 18042, pages 1435-1712; Gombotz and Pettit (1995), Bioconjugate Chem. , .6:332-351; Leone-Bay, et al .
  • hyaluronan is intended to include hyaluronan, hyaluronic acid, salts thereof (such as sodium hyaluronate) , esters, ethers, enzymatic derivatives and cross-linked gels of hyaluronic acid, and chemically modified derivatives of hyaluronic acid (such as hylan) .
  • Exemplary forms of hyaluronan are disclosed in U.S. Patent Nos.
  • hyaluronan Suppliers of hyaluronan include BioMatrix , Inc., Ridgefield, NJ; Fidia S.p.A., Abano Terme, Italy; Kaken Pharmaceutical Co., Ltd., Tokyo, Japan; Pharmacia AB, Sweden; Genzyme Corporation, Cambridge, MA; Pronova Biopolymer, Inc. Portsmouth, NH;
  • a liquid solution or suspension can be used in a manner similar to a mouthwash, where the liquid is swished around in the mouth so as to maximize treatment of lesions (United States Patent 5,102,870, the teachings of which are incorporated by reference) .
  • Longer contact with the mucosai surface can be attained by selecting a suitable vehicle which is capable of coating mucosa.
  • suitable vehicles which is capable of coating mucosa.
  • pectin containing formulations such as OrabaseTM (Colgate-Hoyt Laboratories, Norwood, MA), sucralfate suspensions, Kaopectate and Milk of Magnesia.
  • the formulation can also be a spreadable cream, gel, lotion or ointment having a pharmaceutically acceptable non-toxic vehicle or carrier.
  • KGF protein product (s) can also be incorporated into a slow dissolving lozenge or troche, a chewing gum base, or a buccal or slow delivery prosthesis hooked onto a back molar, for example.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit may each contain both a first container having a dried protein and a second container having an aqueous formulation.
  • Kits included within the scope of this invention are single and multi-chambered pre-filled syringes; exemplary pre-filled syringes (e.g., liquid syringes, and lyosyringes such as Lyo-Ject®, a dual- chamber pre-filled lyosyringe) are available from Vetter GmbH, Ravensburg, Germany.
  • KGF protein product (s) may be applied in therapeutically- and prophylactically-effective amounts to organs or tissues specifically characterized by having damage to or clinically insufficient numbers of epithelium cells. It should be noted that KGF protein product (s) formulations described herein may be used for veterinary as well as human applications and that the term "patient” should not be construed in a limiting manner.
  • the frequency of dosing the KGF protein product (s) to a patient will depend on the disease and the condition of the patient, as well as the pharmacokinetic parameters of KGF protein product (s) as formulated, and the route of administration.
  • the KGF protein product (s) may be administered once, administered daily, or administered with an initial bolus dose followed by a continuous dose or sustained delivery. It is also contemplated that other modes of continuous or near-continuous dosing may be practiced. For example, chemical derivatization may result in sustained release forms of the protein which have the effect of a continuous presence in the bloodstream, in predictable amounts, based on a determined dosage regimen.
  • a patient in need of stimulation (including cytoprotection, proliferation and/or differentiation) of epithelial cells may be administered an effective amount of a KGF protein product (s) to elicit the desired response in the patient.
  • a KGF protein product s
  • the dosage regimen involved in a method of preventing or treating a specific condition generally will be determined by the attending physician, considering various factors which modify the action of drugs, e.g., the age, condition, body weight, sex and diet of the patient, the severity of any infection, the time of administration and other clinical factors. Appropriate dosages may be ascertained through use of established assays for determining dosages utilized in conjunction with appropriate dose-response data.
  • Typical dosages will range from 0.001 mg/kg body weight to 500 mg/kg body weight, preferably up to 200 mg/kg body weight, more preferably 100 mg/kg body weight.
  • the KGF protein product (s) may be administered via topical, enteral or parenteral administration including, without limitation, infusion, intraarterial, intraarticular, intracapsular, intracardiac, intradermal, intramuscular, intraorbital, intrathecal, intravenous, intraperitoneal, intraspinal, intrasternal injection, intraventricular, subcutaneous, subcuticular, subcapsular, subarachnoid and transtracheal .
  • the KGF protein product (s) may be administered via oral administration or administered through mucus membranes, that is, buccally, intranasally, rectally or sublingually for systemic delivery.
  • the KGF protein product (s) may be used once or administered repeatedly, depending on the disease and the condition of the patient. In some cases, the KGF protein product (s) may be administered as an adjunct to other therapy and also with other pharmaceutical preparations.
  • cell therapy is also contemplated, e.g., implantation of cells producing KGF protein(s).
  • This embodiment of the present invention may include implanting into patients cells which are capable of synthesizing and secreting a biologically- active form of KGF protein (s).
  • Such cells producing KGF protein (s) may be cells which do not normally produce KGF protein (s) but which have been modified to produce KGF protein (s) , or which may be cells whose ability to produce KGF protein (s) have been augmented by transformation with a polynucleotide suitable for the expression and secretion of KGF protein(s).
  • the cells be of the same species as the patient (e.g., human), or that the cells may be encapsulated with material that provides a barrier against immune recognition, or that cells be placed into an immunologically-privileged anatomical location, such as in the testis, eye and central nervous system.
  • Human or non-human animal cells may be implanted in patients in biocompatible, semi-permeable polymeric enclosures or membranes to allow release of a KGF protein (s) but prevent destruction of the cells by the patien ' s immune system or by other detrimental factors from the surrounding tissue.
  • the patient's own cells, transformed ex vivo to produce KGF protein(s) could be implanted directly into the patient without such encapsulation.
  • the methodology for the membrane encapsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the encapsulated cells and their implantation in patients may be accomplished with known techniques (U.S. Patent Nos . 4,892,538; 5,011,472; and 5,106,627, the disclosures of which are hereby incorporated by reference) .
  • in vivo gene therapy is also envisioned, wherein a nucleic acid sequence encoding a KGF protein (s) is introduced directly into a patient. Efficient and long lasting gene transfer to hepatocytes is required for effective gene therapy for local expression of the protein to prevent and/or treat liver diseases and/or for secretion of the protein to prevent and/or treat diseases in other organs or tissues.
  • the DNA construct may be directly injected into the tissue of the organ to be treated, where it can be taken up in vivo and expressed, provided that the DNA is operable linked to a promoter that is active in such tissue.
  • the DNA construct may also additionally include vector sequence from such vectors as an adenovirus vector, a retroviral vector, papilloma virus and/or a herpes virus vector, to aid uptake in the cells.
  • Physical transfer may be achieved in vivo by local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, such as liposome-mediated transfer, direct injection (naked DNA) , receptor-mediated transfer (ligand-DNA complex) , or microparticle bombardment (gene gun) .
  • a KGF protein product (s) may be applied in therapeutically- and prophylactically-effective amounts to organs or tissues specifically characterized by having damage to or clinically insufficient numbers of epithelium cells .
  • a KGF protein product (s) may be used for veterinary as well as human applications and that the term "patient” should not be construed in a limiting manner.
  • a KGF protein product (s) may be used in vivo to induce stimulation (including cytoprotection, proliferation and/or differentiation) , proliferation and/or differentiation of epithelial cells including, but not limited to, the eye, ear, gums, hair, lung, skin, pancreas (endocrine and exocrine) , thymus, thyroid, urinary bladder, liver and gastrointestinal tract including cells in the oral cavity, in the esophagus, in the glandular stomach and small intestine, in the colon and the intestinal mucosa, in the rectum and in the anal canal .
  • esophagus, stomach and intestines include erosive gastritis, esophagitis, esophageal reflux or inflammatory bowel diseases, such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel) ; disorders or damage to salivary gland tissue including radiation/chemotherapy effects, autoimmune diseases such as Sjogren's Syndrome which can cause salivary gland insufficiency (sicca syndrome) ; insufficient production of mucus throughout the gastrointestinal tract; adult respiratory distress syndrome (ARDS) , pneumonia, hyaline membrane disease (i.e., infant respiratory distress syndrome and bronchopulmonary dysplasia) in premature infants; acute or chronic lung damage or insufficiency due to inhalation injuries (including high oxygen levels) , emphysema, lung damage from chemotherapeutics, ventilator trauma or other lung damaging circumstances; hepatic cirrhosis, fulminant liver failure, damage caused
  • Therapeutic agents such as analgesics and anesthetics also can be administered to alleviate pain and such as anti-infectictives, anti-bacterials, anti- fungals and/or antiseptics also can be administered to prevent and/or treat secondary infection of the lesions.
  • KGF protein product (s) specifically characterized by the prophylactic and/or therapeutic use of KGF to reduce, delay and/or block the onset of damage to or deficiencies in these particular types of cells.
  • KGF protein product (s) are useful to increase cytoprotection, proliferation and/or differentiation of hepatocytes in order to increase liver function.
  • KGF protein product (s) are useful to treat and/or prevent hepatic cirrhosis, fulminant liver failure, damage caused by acute viral hepatitis, toxic insults to the liver and/or bile duct disorders.
  • Hepatic cirrhosis secondary to viral hepatitis and chronic alcohol ingestion, is a significant cause of morbidity and mortality.
  • KGF protein product (s) are useful to treat and/or prevent the development of cirrhosis.
  • a standard in vivo model of hepatic cirrhosis is known (Tomaszewski et al . (1991), J. Appl . Toxicol . , H:229-231, the disclosure of which is hereby incorporated by reference) .
  • Fulminant liver failure is a life-threatening condition which occurs with end-stage cirrhosis and which is presently treatable only with liver transplantation.
  • KGF protein product (s) are useful to treat and/or prevent fulminant liver failure.
  • Standard in vivo models of fulminant liver failure are known (Mitchell et al . (1973), J. Pharmacol . Exp. Ther . ,
  • Acute viral hepatitis is frequently subclini- cal and self-limiting. However, in a minority of patients severe liver damage can result over several weeks. KGF protein product (s) are useful in preventing and/or treating viral hepatitis. Standard in vivo models of hepatocyte proliferation are known (Housley et al .
  • KGF protein product (s) are useful to increase cytoprotection, proliferation and/or differentiation of epithelial cells in the gastrointestinal tract (e.g., the oral cavity, esophagus, stomach, small intestine, colon, rectum and anal canal) .
  • gastrointestinal tract e.g., the oral cavity, esophagus, stomach, small intestine, colon, rectum and anal canal
  • the terms "gastrointestinal tract”, as defined herein, and “gut” are art-recognized terms and are used interchangeably herein.
  • KGF protein product (s) are useful to treat and/or prevent gastric ulcers, duodenal ulcers, inflammatory bowel disease, gut toxicity and erosions of the gastrointestinal tract.
  • KGF protein product are useful to prevent degeneration of glandular mucosa and to regenerate glandular mucosa more rapidly, e.g., offering a significant therapeutic improvement in the treatment of gastric ulcers.
  • Standard in vivo models of gastric ulcers are known
  • KGF protein product (s) are useful to prevent degeneration of the mucosai lining of the duodenum and to rapidly regenerate the mucosai lining of the duodenum to heal those ulcers and decrease their recurrence.
  • Standard in vivo models of duodenal ulcers are known (Berg et al . (1949), Proc . Soc . Exp . Biol . Med. , 7:374-376; Szabo and Pihan, Chronobiol . Int . (1987), 6:31-42; and Robert et al . (1970), Gastroenterology, 59 . : 95-102, the disclosures of which are hereby incorporated by reference) .
  • Gut toxicity is a major limiting factor associated with cancer treatment, both in radiation (abdominal, total body or local, e.g., head and neck) and chemotherapy.
  • chemotherapy for cancer such as leukemia, breast cancer or as an adjuvant to tumor removal
  • radiotherapy for head and neck cancer and combined chemotherapy and radiotherapy for bone marrow transplants.
  • the severity of damage is related to the type and dose of chemotherapeutic agent (s) and concomitant therapy such as radiotherapy.
  • Mucositis in portions of the gastrointestinal tract may account for significant pain and discomfort for these patients, and range in severity from redness and swelling to frank ulcerative lesions. The lesions often become secondarily infected and become much harder to heal. Standard in vivo models of radiation- induced gut toxicity are known (Withers and Elkind (1970), Int . J. Radiat . , 17 (3) :261-267, the disclosure of which is hereby incorporated by reference) .
  • chemotherapeutic agents include, but are not limited to, BCNU, busulf n, carboplatin, cyclophosphamide, cisplatin, cytosine arabinoside, daunorubicin, doxorubicin, etoposide, 5-fluorouracil, gemcytabine, ifosphamide, irinotecan, melphalan, methotrexate, navelbine, topotecan, taxol and taxotere, and exemplary treatment regimes include, but are not limited to, BEAM (busulfan, etoposide, cytosine arabinoside, methotrexate) ; cyclophosphamide and total body irradiation; cyclophosphamide, total body irradiation and etoposide; cyclophosphamide and busulfan; and 5-fluorouracil with leucovorin or levamisole.
  • BCNU busulf n
  • KGF protein product (s) Treatment, pretreatment and/or post- treatment, with KGF protein product (s) are useful to generate a cytoprotective effect or regeneration or both, for example, on the small intestinal mucosa, allowing increased dosages of such therapies while reducing potential fatal side effects of gut toxicity.
  • KGF protein product (s) may preferentially be administered in the following settings . Colorectal patients routinely are administered 5-fluorouracil with leucovorin on days 1 to 5; KGF protein product (s) may be administered on days -2, -1 and 0.
  • KGF protein product (s) may be administered on days -2, -1 and 0 and thereafter once per week until the end of the radiation therapy.
  • BEAM therapy for 6 days (days 1 to 6); KGF protein product (s) may be administered on days -2, -1 and 0 and as a three day post-treatment (days 7 to 9) .
  • KGF protein product (s) may be administered prophylactically and/or therapeutically to reduce, delay and/or block the onset of mucositis (due to chemotherapy and/or radiotherapy) , in combination with one or more cytokines to delay and/or block the onset of cytopenia.
  • bone marrow, peripheral blood progenitor cells or stem cells are removed from a patient prior to myelosuppressive cytoreductive therapy (chemotherapy alone or with radiation therapy) and are then readministered to the patient concurrent with or following cytoreductive therapy in order to counteract the myelosuppressive effects of such therapy.
  • myelosuppressive cytoreductive therapy chemotherapy alone or with radiation therapy
  • cytoreductive therapy chemotherapy alone or with radiation therapy
  • Many different approaches have been undertaken to protect an organism from the side effects of radiation or toxic chemicals.
  • One approach is to replace bone marrow cells before toxicity has developed.
  • PBPC peripheral blood
  • PBPC peripheral blood
  • G-CSF or GM-CSF cytokine therapy alone
  • chemotherapy or cytokines chemotherapy or cytokines. They can be given back fresh or cryopreserved.
  • the cells may be CD34+ selected, Tn-cell depleted, tumor cell depleted, or the progenitor cells can be expanded (caused to multiply) by means known in the art, prior to administration.
  • the benefits of re-infusion of autologous or allogeneic progenitors following myelosuppressive therapy have been described in the literature (Morse et al . (1992), Ann .
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines and traditional polypeptide hormones. Included among the cytokines are insulin-like growth factors; human growth hormone; N-methionyl human growth hormone; bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH) , thyroid stimulating hormone (TSH) and leutinizing hormone (LH) ; hemopoietic growth factor; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and -beta; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin; nerve growth factors such as NGF-be
  • KGF protein product(s) should be coordinated and optimized.
  • an appropriate dose of KGF protein product (s) can be administered prior to or subsequent to administration of the therapeutic agent (s) .
  • a parameter to be considered is whether the cytokine is administered in a single dose or in multiple doses during the course of therapy. Certain cytokines are cleared rapidly from the body and will require periodic or continuous administration in order for their efficacy to be maximized. The manner of administration can differ, depending on whether a pre- treatment or post-treatment of the cytokine is given.
  • the cytokine is given prior to the cytotoxic agent, it is preferable to administer the cytokine by intravenous bolus injection for several hours and, optionally, to repeat such administration on one or more days during and after completion of the cytotoxic therapy.
  • KGF protein product(s) are administered (e.g., intraveneously) at 0.1 to 500 micrograms/kg/dose, preferably up to about 200 micrograms/kg/dose, prior to (e.g., 1 to 3 days) and/or after chemotherapy or radiation therapy, and G-CSF (NeupogenTM or LenograstimTM) or GM-CSF (SargramostimTM) is administered (e.g., subcutaneously) at 5 micrograms/kg/dose for 1 to 10 days (preferably 7 to 10 days) after chemotherapy.
  • Erosions of the gastrointestinal tract include erosive gastritis, esophagitis, esophageal reflux and inflammatory bowel diseases.
  • Inflammatory bowel diseases such as Crohn ' s disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel) , are chronic diseases of unknown etiology which result in the destruction of the mucosai surface, inflammation, scar and adhesion formation during repair, and significant morbidity to the affected individuals.
  • KGF protein product (s) are useful to regenerate the mucosai lining and decrease the recurrence of these erosions, resulting in faster healing, and may be of benefit in controlling progression of the disease.
  • Standard in vivo models of erosion of the gastrointestinal tract are known (Geisinger et al . (1990), Mod-Pa thol . , 3 (5) :619-624; Carlborg et al . (1983), Laryngoscope, 93 (2 ) :184-187; Carlborg et al . (1980), Eur-Surg-Res . , 12 (4) :270-282; Keshavarzian et al . (1991), Alcohol -Clin-Exp-Res . , 15(1) ; 116-121; Katz et al . (1988), Dig-Dis-Sci . , Hi l: 217-224; and Zeeh et al . (1996),
  • Gastroenterology 110 (4) :1077-1083 , the disclosures of which are hereby incorporated by reference) .
  • Standard in vivo models of inflammatory bowel disease are well known (Morris et al . (1989), Gastroenterology, 96 :795- 803; Rachmilewitz et al . (1989), Gastroenterology, 97:326-327; Allgayer et al . (1989), Gastroenterology, 16:1290-1300; and Kim and Borstad (1992), Scand. J. Gastroenterol , 27 (7) :529-537, the disclosures of which are hereby incorporated by reference) .
  • TPN total parenteral nutrition
  • intestinal mucosai atrophy Animal studies have established the relationship between total parenteral nutrition (TPN) and intestinal mucosai atrophy (Buchman et al . (1995), Journal of Parenteral and Enter al Nutri tion, 19 : 453- 460) .
  • the decrease in intestinal villus height is attributed to the lack of growth stimulus provided through oral intake of nutrients. This is reflected in a reduction in the labeling index, a measure of growth. Decreases in villus height are also correlated with decreases in specific activities of enzymes involved in nutrient absorption.
  • KGF protein product (s) are useful to either protect against atrophy during the fasting and/or facilitate regrowth upon reintroduction of oral nutrients.
  • the present invention is directed to the method of using KGF protein products and GLP-2 protein products in combination to increase cytoprotection, proliferation and/or differentiation of epithelial cells in the gastrointestinal tract.
  • the combination therapy is useful to treat and/or prevent gastric ulcers, duodenal ulcers, inflammatory bowel disease, gut toxicity and erosions of the gastrointestinal tract.
  • GLP-2 peptide refers collectively herein to human GLP-2 (SEQ ID NO: 67) and to variants of thereof (defined, for example, in WO 96/32414 and, for example, hGLP-2(G2), a variant GLP-2 in which glycine is substituted for asparagine at position 2 (SEQ ID NO:68) and hGLP-2(S2), a variant GLP-2 in which serine is substituted for asparagine at position 2 (SEQ ID NO:69), described herein).
  • GLP-2 and variants thereof may be produced and purified by standard techniques (see, for example, WO 96/32414, the disclosure of which is hereby incorporated by reference) .
  • WO 96/32414 reported that GLP-2 has effects on small bowel and pancreatic islets.
  • Hyaline membrane disease of premature infants results in the absence of surfactant production by type II pneumocytes within the lung, resulting in the collapse of the alveoli.
  • KGF protein product (s) are useful to treat and/or prevent hyaline membrane disease.
  • Smoke inhalation is a significant cause of morbidity and mortality in the week following a burn injury, due to necrosis of the bronchiolar epithelium and the alveoli.
  • KGF protein products are useful treat and/or prevent inhalation injuries.
  • Emphysema results from the progressive loss of alveoli.
  • KGF protein product (s) are useful to treat and/or prevent emphysema.
  • Disorders of the pancreas may be endocrine- related such as Type I or Type II diabetes, or may be exocrine-related such as pancreatitis and pancreatic inefficiencies or cystic fibrosis.
  • Patients with diagnosed Type I diabetes require constant exogenous insulin administration.
  • Patients with diagnosed Type II diabetes progress through varying stages of insulin resistance/insufficiency to ultimately also require exogenous insulin administration.
  • KGF protein product are useful to ameliorate, delay and/or circumvent permanent manifestation of diabetes mellitus or as an adjunct in the setting of islet cell transplantation by inducing pancreatic beta cell function in order to normalize blood glucose levels during varying metabolic demands, yet avoid frequent or profound hypoglycemia.
  • Standard models of diabetes are known (Junod et al . (1967), Proc . Soc . Exp. Bio . Med. 126(1) :201-205; Rerup (1970), Phar . Rev. , 22:485-518; Rossini et al . (1977), P. N. A . S.
  • pancreas 1:50-57, the disclosures of which are hereby incorporated by reference
  • a standard model of pancreatic cell proliferation is known (Yi et al . (1994), American Journal of Pathology, 145 (1) : 80-85, the disclosure of which is hereby incorporated by reference) .
  • Corneal cells may be damaged by corneal abrasion and/or corneal ulcerations due to chemicals, bacteria or viruses.
  • KGF protein product (s) are useful treat and/or prevent corneal degeneration. Standard in vivo models of corneal cell regeneration are known
  • KGF protein product (s) are useful to treat and/or prevent gum disease. Standard in vivo models of gum disease are known.
  • KGF protein product (s) are useful to treat and/or prevent ulcerating and/or inflammatory conditions including conditions related to chemotherapy (as discussed above) and/or infection.
  • Standard in vivo models of urinary bladder damage are known (Ford and Hess (1976), Arch . Intern . Med. , 136:616-619 and Droller, et al . (1982), Urol . , 20:256-258, the disclosures of which are hereby incorporated by reference) .
  • KGF protein product (s) are useful to treat and/or prevent eardrum damage.
  • Standard in vivo models of tympanic membrane perforations are known (Clymer et al. (1996), Laryngoscope (USA) , 106(3) :280-285, the disclosure of which is hereby incorporated by reference) .
  • KGF protein product are useful to treat and/or prevent disorders or damage to salivary gland tissue, including radiation/chemotherapy effects (as discussed above) and autoimmune diseases such as Sjogren's Syndrome which can cause salivary glandinsufficiency (sicca syndrome) . Standard in vivo models of salivary gland tissue damage are known.
  • test materials used in the following in vivo studies were a KGF having a deletion of the first 23 amino acids of the amino-terminus of KGF (i.e., ⁇ N23 KGF) using standard techniques (WO 96/11949) . All proteins were produced by recombinant expression in E. coli and purified to homogeneity. Each protein was administered as a subcutaneous formulation.
  • hGLP-2(G2) polypeptide was synthesized by the Fmoc
  • peptide synthesizer utilizing a single coupling program was used to carry out the chain assembly.
  • Commercially available preloaded Fmoc-amino acid-HMP (hydroxymethyl-phenylacetyl) derivatized polystyrene resin was used to prepare the carboxy- terminal acid peptides .
  • Subsequent amino acids were coupled as HOBT (hydroxybenztriazole) esters.
  • Mass spectra for synthetic peptides were obtained on a Sciex API (Perkin Elmer Corp.) single quadropole mass spectrometer. All mass spectral samples were fractions off of the preparative HPLC purification.
  • mice Normal BDFl mice were treated with ⁇ N23 KGF given as a single daily injection of 5 mg/kg/day alone, hGLP- 2(G2) given as a twice daily injection of 30 ug/injection, or both.
  • the mice were treated for 6 days and euthanized for removal of the intestines on day 7.
  • the entire length of the small intestine was removed, and hung under 5 g tension for measurement of length and subdivision into 3 segments.
  • the first 10 cm was designated duodenum, the last 10 cm designated ileum and the remaining central portion jejunum.
  • the segments were flushed with ice cold saline, patted dry and weighed.
  • the mean wet weight values were converted to percent increases relative to control and the data is presented in Table 2 below.
  • mice (15/group) treated with 5-fluorouracil (Roche Laboratories, Nutley NJ) , were administered with vehicle alone (saline) , and with ⁇ N23 KGF (formulated in saline and/or hGLP-2 (G2) in 0.1M ammonium bicarbonate, according to the following schedule:
  • Control group On days -2 to 0, a group of mice were administered saline on days -2 to 0, and injected intraperitoneally with 5-fluorouracil (5-FU, 60 mg/kg/day) on days 1 to 4.
  • 5-fluorouracil 5-FU, 60 mg/kg/day
  • ⁇ N23 KGF pretreatment On days -2 to 0, a group of mice were injected subcutaneously with ⁇ N23 KGF (5 mg/kg) . On days 1 to 4, the mice were injected intraperitoneally with 5-fluorouracil (5-FU, 60 mg/kg/day) .
  • hGLP-2 (G2) pretreatment On days -5 to 0, a group of mice were injected subcutaneously with hGLP-2 (G2) (3 mg/kg (split dose)) . On days 1 to 4, the mice were injected intraperitoneally with 5-fluorouracil (5- FU, 60 mg/kg/day) .
  • ⁇ N23 KGF and hGLP-2 (G2) pretreatment On days -2 to 0, a group of mice were injected subcutaneously with hGLP-2 (G2) (3 mg/kg (split dose)) and injected subcutaneously with ⁇ N23 KGF (5 mg/kg) . On days 1 to 4, the mice were injected intraperitoneally with 5-fluorouracil (5-FU, 60 mg/kg/day) .
  • pretreatment with hGLP- 2(G2) administered alone did not protect against weight loss, but pretreatment with ⁇ N23 KGF administered alone had a significant effect.
  • Pretreatment with ⁇ N23 KGF and hGLP-2 (G2) showed an improvement relative to pretreatment with ⁇ N23 KGF or hGLP-2 (G2) alone.
  • Hepatic abscesses are commonly found in control, but not ⁇ N23 KGF-pretreated, surviving mice indicating that 5-FU's toxicity is in part due to loss of the gastrointestinal barrier function.
  • ⁇ N23 KGF-pretreated mice lose less weight and consume more food and water during the 5-FU treatment period.
  • Pretreatment with ⁇ N23 KGF increased survivability at 30 days relative to saline (75% vs. 35%), but not concurrent treatment (0% vs. 35%).
  • Pretreatment with ⁇ N23 KGF and hGLP-2 (G2) resulted in all animals survived for 30 days.
  • hGLP-2 (G2) given concurrently with ⁇ N23 KGF as a pretreatment significantly improved survival compared to survival in group given ⁇ N23 KGF alone as a pretreatment.
  • Trp lie Leu Thr Trp lie Leu Pro Thr Leu Leu Tyr Arg Ser Cys Phe 5 10 15
  • AGT 452 lie Arg Thr Val Ala Val Gly He Val Ala He Lys Gly Val Glu Ser 100 105 110 115
  • MOLECULE TYPE protein
  • SEQUENCE DESCRIPTION SEQ ID NO : 4 :
  • GCA TCT GCT AAA TGG ACC CAC AAC GGT GGT GAA ATG TTC GTT GCT CTG 384 Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val Ala Leu 115 120 125
  • TCT GCT AAA TGG ACC CAC AAC GGT GGT GAA ATG TTC GTT GCT CTG AAC 384 Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val Ala Leu Asn 115 120 125
  • GGT GAA ATG TTC GTT GCT CTG AAC CAG AAA
  • GGT ATC CCG GTT CGT GGT 384
  • Gly Glu Met Phe Val Ala Leu Asn Gin Lys Gly He Pro Val Arg Gly 115 120 125
  • MOLECULE TYPE protein
  • AAG GGC ACC CAA GAG ATG AAA AAC AAC TAC AAT ATT ATG GAA ATC CGT 144 Lys Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn He Met Glu He Arg 35 40 45
  • GGC ACC CAA GAG ATG AAA AAC AAC TAC AAT ATT ATG GAA ATC CGT ACT 144 Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn He Met Glu He Arg Thr 35 40 45
  • MOLECULE TYPE protein
  • AAG GGC ACC CAA GAG ATG AAA AAC AAC TAC AAT ATT ATG GAA ATC CGT 144 Lys Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn He Met Glu He Arg 35 40 45
  • AGA GGA AAA AAA ACG AAG AAA GAA CAA AAA ACA GCC CAC TTT CTT CCT 480 Arg Gly Lys Lys Thr Lys Lys Glu Gin Lys Thr Ala His Phe Leu Pro 145 150 155 160

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Cosmetics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP97953157A 1996-12-06 1997-12-08 Verwendung eines kgf-proteinproduktes und eines glp-2 proteinproduktes für die herstellung eines medikamentes Expired - Lifetime EP1012186B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
SI9730362T SI1012186T1 (en) 1996-12-06 1997-12-08 Use of a kgf protein product(s) and a glp-2 protein product(s) for the preparation of a medicament

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US3253396P 1996-12-06 1996-12-06
US32533P 1996-12-06
US6207497P 1997-10-15 1997-10-15
US62074P 1997-10-15
PCT/US1997/022735 WO1998024813A2 (en) 1996-12-06 1997-12-08 Keratinocyte growth factors and their use in combination with glucagon-like peptide derivatives

Publications (2)

Publication Number Publication Date
EP1012186A2 true EP1012186A2 (de) 2000-06-28
EP1012186B1 EP1012186B1 (de) 2002-07-17

Family

ID=26708564

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97953157A Expired - Lifetime EP1012186B1 (de) 1996-12-06 1997-12-08 Verwendung eines kgf-proteinproduktes und eines glp-2 proteinproduktes für die herstellung eines medikamentes

Country Status (10)

Country Link
EP (1) EP1012186B1 (de)
JP (1) JP4091988B2 (de)
AT (1) ATE220689T1 (de)
AU (1) AU5696298A (de)
CA (1) CA2272854C (de)
DE (1) DE69714085T2 (de)
DK (1) DK1012186T3 (de)
ES (1) ES2181054T3 (de)
PT (1) PT1012186E (de)
WO (1) WO1998024813A2 (de)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6743422B1 (en) 1996-10-15 2004-06-01 Amgen, Inc. Keratinocyte growth factor-2 products
CA2289652C (en) * 1997-05-16 2012-02-28 1149336 Ontario Inc. Methods of enhancing functioning of the upper gastrointestinal tract
US6248725B1 (en) 1999-02-23 2001-06-19 Amgen, Inc. Combinations and methods for promoting in vivo liver cell proliferation and enhancing in vivo liver-directed gene transduction
US7186683B2 (en) 2000-09-18 2007-03-06 Sanos Bioscience A/S Use of GLP for the treatment, prevention, diagnosis, and prognosis of bone-related and nutrition-related disorders
WO2002022151A2 (en) 2000-09-18 2002-03-21 Osteometer Bio Tech A/S Use of glp-1 and flp-2 peptides for treatment of bone disorders
US7371721B2 (en) 2000-09-18 2008-05-13 Sanos Bioscience A/S Use of GLP-2 and related compounds for the treatment, prevention, diagnosis, and prognosis of bone-related disorders and calcium homeostasis related syndromes
CA2436399A1 (en) 2001-02-16 2002-08-29 Conjuchem Inc. Long lasting glucagon-like peptide 2 (glp-2) for the treatment of gastrointestinal diseases and disorders
MXPA04001526A (es) 2001-08-21 2004-05-31 Chiron Corp Composiciones de polipetidos kgf.
AU2007214362B2 (en) * 2001-08-21 2009-11-26 Novartis Vaccines And Diagnostics, Inc. KGF polypeptide compositions
US8304387B2 (en) * 2004-12-15 2012-11-06 Swedish Orphan Biovitrum Ab (Publ) Therapeutic formulations of keratinocyte growth factor
WO2015063613A2 (en) 2013-11-01 2015-05-07 Spherium Biomed S.L. Inclusion bodies for transdermal delivery of therapeutic and cosmetic agents
EP4043010A1 (de) * 2016-05-06 2022-08-17 Phasebio Pharmaceuticals, Inc. Elp-fusionsproteine zur gesteuerten und verzögerten freisetzung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9824813A2 *

Also Published As

Publication number Publication date
JP2001510333A (ja) 2001-07-31
JP4091988B2 (ja) 2008-05-28
DE69714085T2 (de) 2002-11-14
EP1012186B1 (de) 2002-07-17
ATE220689T1 (de) 2002-08-15
WO1998024813A3 (en) 1998-08-06
DK1012186T3 (da) 2002-11-04
PT1012186E (pt) 2002-12-31
DE69714085D1 (de) 2002-08-22
AU5696298A (en) 1998-06-29
CA2272854C (en) 2004-02-10
WO1998024813A2 (en) 1998-06-11
ES2181054T3 (es) 2003-02-16
CA2272854A1 (en) 1998-06-11

Similar Documents

Publication Publication Date Title
JP2008283980A (ja) ケラチノサイト増殖因子−２産物
AU774307B2 (en) Truncated soluble tumor necrosis factor type-1 and type-11 receptors
EP0804479B1 (de) Verfahren zur behandlung von diabetes mittels kgf
TWI351966B (en) Therapeutic formulations of keratinocyte growth fa
CA2272854C (en) Keratinocyte growth factors and their use in combination with glucagon-like peptide derivatives
AU725509B2 (en) Uses of keratinocyte growth factor-2
US6743422B1 (en) Keratinocyte growth factor-2 products
EP1452205B1 (de) Verwendung von keratinocyte growth factor-2
US20030144202A1 (en) Uses of keratinocyte growth factor-2
AU775027B2 (en) Keratinocyte growth factor-2 products
EP1473366A1 (de) Produkte des Keratinocyten-Wachstumsfaktors 2
MXPA99005163A (en) Keratinocyte growth factors and uses thereof
CA2452540A1 (en) Keratinocyte growth factor-2 products
MXPA00002762A (en) Keratinocyte growth factor-2 products
MXPA99003532A (en) Keratinocyte growth factor-2 products
MXPA99003528A (en) Uses of keratinocyte growth factor-2

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990706

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL PAYMENT 19990706;LT PAYMENT 19990706;LV PAYMENT 19990706;MK PAYMENT 19990706;RO PAYMENT 19990706;SI PAYMENT 19990706

17Q First examination report despatched

Effective date: 20000728

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

RTI1 Title (correction)

Free format text: USE OF A KGF PROTEIN PRODUCT(S) AND A GLP-2 PROTEIN PRODUCT(S) FOR THE PREPARATION OF A MEDICAMENT

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL PAYMENT 19990706;LT PAYMENT 19990706;LV PAYMENT 19990706;MK PAYMENT 19990706;RO PAYMENT 19990706;SI PAYMENT 19990706

REF Corresponds to:

Ref document number: 220689

Country of ref document: AT

Date of ref document: 20020815

Kind code of ref document: T

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: KIRKER & CIE SA

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REF Corresponds to:

Ref document number: 69714085

Country of ref document: DE

Date of ref document: 20020822

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20020403135

Country of ref document: GR

ET Fr: translation filed
REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20021016

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2181054

Country of ref document: ES

Kind code of ref document: T3

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20030422

REG Reference to a national code

Ref country code: SI

Ref legal event code: IF

REG Reference to a national code

Ref country code: PT

Ref legal event code: PC4A

Owner name: BIOVITRUM AB (PUBL), SE

Effective date: 20090528

REG Reference to a national code

Ref country code: CH

Ref legal event code: PUE

Owner name: BIOVITRUM AB (PUBL)

Free format text: AMGEN INC.,#ONE AMGEN CENTER DRIVE#THOUSAND OAKS, CALIFORNIA 91320-1799 (US) -TRANSFER TO- BIOVITRUM AB (PUBL)# #112 76 STOCKHOLM (SE)

Ref country code: CH

Ref legal event code: NV

Representative=s name: BUGNION S.A.

REG Reference to a national code

Ref country code: GB

Ref legal event code: 732E

Free format text: REGISTERED BETWEEN 20090709 AND 20090715

REG Reference to a national code

Ref country code: SI

Ref legal event code: SP73

Owner name: BIOVITRUM AB (PUBL); SE

Effective date: 20090624

REG Reference to a national code

Ref country code: FR

Ref legal event code: TP

REG Reference to a national code

Ref country code: NL

Ref legal event code: SD

Effective date: 20110120

REG Reference to a national code

Ref country code: ES

Ref legal event code: PC2A

Owner name: BIOVITRUM AB (PUBL)

Effective date: 20111018

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DK

Payment date: 20141204

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: MC

Payment date: 20141205

Year of fee payment: 18

Ref country code: GB

Payment date: 20141205

Year of fee payment: 18

Ref country code: CH

Payment date: 20141204

Year of fee payment: 18

Ref country code: IE

Payment date: 20141203

Year of fee payment: 18

Ref country code: LU

Payment date: 20141204

Year of fee payment: 18

Ref country code: ES

Payment date: 20141215

Year of fee payment: 18

Ref country code: GR

Payment date: 20141202

Year of fee payment: 18

Ref country code: FI

Payment date: 20141204

Year of fee payment: 18

Ref country code: SE

Payment date: 20141218

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: AT

Payment date: 20141203

Year of fee payment: 18

Ref country code: NL

Payment date: 20141202

Year of fee payment: 18

Ref country code: PT

Payment date: 20141202

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20141216

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20141212

Year of fee payment: 18

Ref country code: DE

Payment date: 20141203

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20141212

Year of fee payment: 18

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

REG Reference to a national code

Ref country code: PT

Ref legal event code: MM4A

Free format text: LAPSE DUE TO NON-PAYMENT OF FEES

Effective date: 20160608

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 69714085

Country of ref document: DE

REG Reference to a national code

Ref country code: LT

Ref legal event code: MM9D

Effective date: 20151208

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20151231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 220689

Country of ref document: AT

Kind code of ref document: T

Effective date: 20151208

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20151208

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160608

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151209

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20160101

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20160831

Ref country code: SI

Ref legal event code: KO00

Effective date: 20160830

REG Reference to a national code

Ref country code: GR

Ref legal event code: ML

Ref document number: 20020403135

Country of ref document: GR

Effective date: 20160707

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160101

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160701

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160707

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20170127

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151209

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20151208